N-linked Glycosylation Modification And Expression Of Human Proinsulin Gene

Insect baculovirus expression system is the commonly used expression system ofrecombinant proteins, As a eukaryote expression system, the most important feature is totranslate the protein modification, Such as glycosylation, phosphorylation, acetylation,methylation. Modified sites is similar with natural proteins in the cells. This is particularsignificance for many glycoprotein correct folding and biological functions.Human insulin is the non-glycosylation protein hormone, it has a very short half-life invivo. N-glycosylation of Protein has a significant role in its correct folding in thephysiological functions and the biological half-life extension. This study was conducted onthe basis of existing investigations of insulin analogues, conbining with technology ofglycobiology and proinsulin human DNA was reformed.conservative N-glycosylation wasadded to B band terminal，which made the expression in Eukaryotic expression system thathad been modified to be possible and Sugar chain was generated on the Transformationpoint which can prolong the half-life of human proinsulin without biological function beingaffected and causing the immune response. The results showed that after N-glycosylationmodified human proinsulin was expressed in insect cells and silkworm, and its molecularweight is slightly bigger than the non-modification glycosylated human proinsulin protein.Tunicamycin experiments showed that human proinsulin was glycosylation in Bombyx moricells. This study further expressioned the human proinsulin with glycosylation sites inprokaryotes that Escherichia coli, then compared with the eukaryotic expression product.Western Blot showed that the molecular weight of Bombyx mori cells is bigger than in theEscherichia coli, so as to further illustrate that with glycosylation sites of human proinsulinin insect cells inside the expression and glycosylation.In this study, human proinsulin gene was genetically engineered, added-glycosylationconservative base sequence in the destination sites, and glycosylation of the human roinsulinprotein expressed by the baculovirus expression system. Explore the feasibility of using thebaculovirus expression system to produce long-acting human insulin.